My ultimate goal is to become a fully independent principal investigator with a basic science laboratory examining the molecular physiology of renal ion transport. After completing 6 years of training with Dr. Steven Herbert at 3 different institutions, I became an Assistant Professor at the University of Chicago. I have studied the regulation of the rat thiazide-sensitive sodium chloride cotransporter (rNCC) by glycosylation and phosphorylation for that period of time. I have demonstrated that rNCC is capable of being PKC phosphorylated in vitro, rNCC is functionally inhibited by PKC and there is a significant increase in baseline function and surface expression of rNCC with mutation of consensus PKC phosphorylation sites. With the support of this Career Development Award, the University of Chicago Department of Medicine, and my mentors, Dr. Herbert and Dr. Chang, I hope to continue to progress towards my long term goal. NCC is the site of action of one of the most commonly prescribed anti-hypertensive medications and plays a key role in divalent cation handling, yet we know little about it's regulation. This proposal will test the hypothesis that PKC and WNK4 kinase play a key role in regulation of NCC, predominantly by phosphorylation of serine and threonine residues resulting in decreased surface expression. The first specific aim will seek to define the role and mechanisms of WNK4 kinase, PKC and PKA in regulating the activity and surface expression of NCC. The second specific aim will seek to explore the physiological and hormonal regulation of the cotransporter and kinases, the physiological role of the cotransporter in divalent ion handling and the mechanisms underlying these processes. This investigation of the regulation of this effector of blood pressure and calcium homeostasis will provide invaluable insight into the pathogenesis of dysregulation of these homeostatic mechanisms.